Electrophoresis, the process whereby charged molecules migrate in an electric field, is often used to separate mixtures of peptides, proteins or nucleic acids. Electrophoresis of proteins is generally carried out in gels made up of the cross-linked polymer polyacrylamide. Samples containing proteins are loaded in wells or depressions at the top of the polyacrylamide gel and the proteins contained in each sample move into the gel in separate lanes when an electric field is applied. The polyacrylamide gel acts as a molecular sieve, slowing the migration of the proteins approximately in proportion to their size, or molecular weight. Polyacrylamide is often used as the gel matrix for short DNAs (up to a few hundred molecules), and agarose is generally used as the gel matrix for separating longer DNA molecules up to the size of the entire chromosomes.
The most common technique for detection of molecules in a slab gel during electrophoresis comprises exciting them with a laser of a suitable wavelength, collecting the ensuing fluorescence and measuring its intensity (Regnier, F. E., He, B., Lin, S. and Busse, J. (1999), “Chromatography and electrophoresis on chips: critical elements of future integrated, microfluidic analytical systems for life sciences”, Trends Biotech. 17:101-106). In order to use this technique, the electrophoresed molecules should be either capable of emitting fluorescence efficiently, or labeled by another molecule which can do so. While the laser provides a collimated beam of light, a great deal of dispersion or diffraction occurs once within the gel, and therefore the effective cross-section of the beam increases with penetration into the gel to a size considerably larger than at entry thereto, and the intensity is correspondingly reduced. Thus, in order to enable each successive species of molecule that migrates across the laser path to be detected by interaction therewith, it must be ensured that only one such species of molecule at any one time is within the expanded cross-section of the laser beam. Thus, the excitation of the molecules must be conducted at a certain distance from the origin such that the different molecules have already achieved sufficient separation from each other, so that only one type of molecule interacts with the laser beam at any one time. In consequence, the migration length, and therefore the length of the slab gel must be sufficiently long to provide the required separation, which may run, for example, to at least 30 to 50 cm, depending on the gel.
Thus, when the electrophoresis apparatus is adapted for a large slab gel, as in some current DNA sequencing machines, such as in the ALFexpress DNA Analyser® (Amersham Pharmacia Biotech, Uppsala, Sweden), a fixed laser beam is directed into the gel through its side, perpendicular to the direction of the band migration, which excites the fluorescently labeled DNA bands. The resulting fluorescence is then detected by a series of photodiodes (each for each lane) located behind the gel at a right angle to the exciting beam (Sequencing Handbook, (2000), Amersham Pharmacia Biotech, Uppsala, Sweden).
Thus, in the prior art, in order to overcome the problem of laser diffraction within the gel, a rather large sample (having sufficient molecules that are to be detected) must be used, coupled with a sufficiently long gel length to enable the required resolution to be achieved.
However, when the electrophoresis apparatus is a very narrow microfabricated capillary with a cross section of a few tens of micrometers, and the number of electrophoresed molecules is extremely small (typically at the nM range), both the excitation and collection of the emitted photons need to be focused into a tiny volume to minimize background fluorescence and to achieve the required resolution (Mathies, R. A. and Huang, X. C. (1992), “Capillary array electrophoresis: an approach to high-speed, high throughput DNA sequencing”, Nature 359:167-169). One approach to achieve this has been to employ an external confocal microscope setup, which is placed close to the electrophoresis apparatus (Regnier, F. E., et al, supra). In recent years, the components of capillary electrophoresis (i.e. buffer and sample reservoirs, capillaries, electrodes) have been constantly miniaturized well into the realm of microelectronics (Colyer, C. L., Tang, T., Chiem, N. and Harrison, D. J. (1997), “Clinical potential of microchip capillary electrophoresis systems”, Electrophoresis 18:1733-1741). Capillary array electrophoresis chips (CAE) which measure 50 mm×75 mm can accommodate many independent microfabricated capillaries and their injection systems (Woolley. A. T. and Mathias, R. A. (1995) “Ultra-high-speed DNA sequencing using capillary electrophoresis chips”, Anal. Chem. 67:3676-3680.). However, it has been so far impossible to integrate the optical detection system on to the CAE chip, and it has remained a bulky and expensive assembly of external lasers and optical systems (Mathias, R. A., Glazer, A. N., Lao, K. and Wooley, A. T., “Electrochemical detector integrated on microfabricated capillary electrophoresis chips”, 1999, The Regents of the University of California, USA). Thus, while this system enables small sample volumes to be used and with relatively short gel lengths, the detection apparatus is large, bulky and relatively expensive and complex.
An alternative method to the above detection scheme is the electrochemical detection of molecules which can be readily oxidized or reduced by electrodes, said electrodes being integrated within an electrophoresis capillary or placed in a close proximity to its end. In comparison with fluorescence detection, electrochemical detection allows for the integration of capillaries and detectors. Electrochemical detection is also very sensitive and can measure quantities as low as 10−15 mole (Takenaka, S., Uto, Y., Kondo, H., Hiara, T. and Takagi, M. (1994) “Electrochemically active DNA probes: detection of target DNA sequences at femtomole level by high-performance liquid chromatography with electrochemical detection”, Anal Biochem. 218:436-443). However, this technique is still not widely used, probably because of difficulties in the detection of small currents or voltages in a capillary, which is subjected to many kV during electrophoresis, and also because of the need for very accurate placement and alignment of the electrodes.
Thus, in the prior art, either long gel lengths are required to provide the required resolution, or, alternatively, when short gel lengths are possible, these systems nevertheless require cumbersome and expensive confocal microscopy devices for detection.